The first component of complement is made up of three distinct plasma glycoproteins, Clq, Clr and Cls. These glycoproteins form a macromolecular calcium ion dependent complex with a molar ratio of 1:2:2. This complex circulates in vivo as a proenzyme which is activated on binding to immune aggregates or insolubilized IgG.
Purification of the three proteins Clq, Clr and Cls has been accomplished in the past by a series of steps which include precipitation of Cl complex at low ionic strengths or by polyethylene glycol, disassociation of Cl in the presence of ethylene diamine tetraacetic acid and resolution of the Cl subcomponents by column chromatography. Final purifications are then carried out. Other procedures include absorption of Cl to immune precipitates followed by elution and subsequent resolution by ion exchange chromatography. It has previously been demonstrated that Clr and Cls subcomponents can be dissociated from Clq by ethylenediamine tetraacetic acid (EDTA) when the Cl complex is bound to IgG-Sepharose resin, (S. N. Assimeh, D. H. Bing and R. H. Painter, J. Immunol. 113, 225-234 (1974); D. H. Bing, J. M. Andrews, F. L. Suddath and R. Spencer, in "Protides of Biological Fluids," H. Peeters, ed., Pergamon Press, New York, 1975, pp. 551-557; W. P. Kolb, L. M. Kolb and E. R. Podack, J. Immunol. 122, 2103-2111 (1979); K. Takahashi, S. Nagasawa and J. Koyama, FEBS Letters 55, 156-160 (1975)). Improved resolution occurred when the IgG was separated from the resin by a spacer which was a p-benzamidoethylamine linkage.
The cold water-ethanol process, or Cohn Process, for the purification of plasma proteins is used widely in the preparation of serum albumin and immune globulins. There have been reports in which Cohn Fractions have been used as starting material for the preparation of a number of materials; however, Cohn Fractions have been found of little use as a starting material for the preparation of highly purified, functionally active complement proteins, perhaps due to the instability of this group of proteins to the conditions used during fractionation. Strong, (L. E. Strong, in "Encyclopedia of Chemical Technology," Vol. 2, R. E. Kirk and D. F. Othmar, eds., Interscience Encyclopedia, New York, 1948, pp. 1-29), in his review of Cohn methodology, indicated that Cl could be found in Fraction III and recently, Granier et al, (C. Granier, A. Faure, R. Tavernier and M. Steinbuch, Prep. Biochem. 9, 281-291 [1979]), reported the isolation of Clq from the euglobulins in this fraction. Furthermore, Lepow and Ratnoff, (O. D. Ratnoff and I. H. Lepow, J. Exp. Med. 106, 327-343 [1957]), suggested that Fractions I and III were a source of the proenzyme form of Cl.
Cohn Fraction I has not normally been used for commercial preparations in the past. In fact, the material is often considered a waste material suitable only for research use.
Yet, it has been difficult to obtain highly purified and active subcomponents of the first component of complement Clq, Clr and Cls which are known to be useful in the clinical testing for immune complexes or investigations of the biochemical activities of complement proteins in plasma or serum.